Actinorhizal signaling molecules : Frankia root hair deforming factor shares properties with NIN inducing factor Maimouna Cissoko, Valérie Hocher, Hassen Gherbi, Djamel Gully, Alyssa Carré-Mlouka, Seyni Sane, Sarah Pignoly, Antony Champion, Mariama Ngom, Petar Pujic, et al.

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Maimouna Cissoko, Valérie Hocher, Hassen Gherbi, Djamel Gully, Alyssa Carré-Mlouka, et al.. Acti- norhizal signaling molecules : Frankia root hair deforming factor shares properties with NIN inducing factor. Frontiers in Plant Science, Frontiers, 2018, 9, 12 p. ￿10.3389/fpls.2018.01494￿. ￿hal-01956101￿

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ORIGINAL RESEARCH published: 18 October 2018 doi: 10.3389/fpls.2018.01494

Actinorhizal Signaling Molecules: Frankia Root Hair Deforming Factor Shares Properties With NIN Inducing Factor

Maimouna Cissoko1,2,3,4, Valérie Hocher4, Hassen Gherbi4, Djamel Gully4, Alyssa Carré-Mlouka4,5, Seyni Sane6, Sarah Pignoly1,2,4, Antony Champion1,2,7, Mariama Ngom1,2, Petar Pujic8, Pascale Fournier8, Maher Gtari9, Erik Swanson10, Céline Pesce10, Louis S. Tisa10, Mame Oureye Sy3 and Sergio Svistoonoff1,2,4*

1 Laboratoire Commun de Microbiologie, Institut de Recherche pour le Développement/Institut Sénégalais de Recherches Agricoles/Université Cheikh Anta Diop, Centre de Recherche de Bel Air, Dakar, Senegal, 2 Laboratoire Mixte International Adaptation des Plantes et Microorganismes Associés Aux Stress Environnementaux, Centre de Recherche de Bel Air, Dakar, Senegal, 3 Laboratoire Campus de Biotechnologies Végétales, Département de Biologie Végétale, Faculté des Sciences et Techniques, Université Cheikh Anta Diop, Dakar, Senegal, 4 Laboratoire des Symbioses Tropicales et Méditerranéennes, Institut de Recherche pour le Développement/INRA/CIRAD, Université Montpellier/SupAgro, Montpellier, France, 5 UMR 7245, Molécules de Communication et Adaptation des Microorganismes, Muséum National d’Histoire Naturelle, Centre National de la Recherche Scientifique, Sorbonne Universités, Paris, France, 6 Laboratoire de Botanique et de Biodiversité Végétale, Département de Biologie Végétale, Faculté des Sciences et Techniques, Université Cheikh Anta Diop, Dakar, Edited by: Senegal, 7 UMR Diversité Adaptation et Développement des Plantes (DIADE), Institut de Recherche pour le Développement, Ulrike Mathesius, Montpellier, France, 8 Ecologie Microbienne, UMR 5557 CNRS, Université Lyon 1, Villeurbanne, France, 9 Institut National Australian National University, des Sciences Appliquées et de Technologie, Université Carthage, Tunis, Tunisia, 10 Department of Molecular, Cellular, Australia and Biomedical Sciences, University of New Hampshire, Durham, NH, United States Reviewed by: Dugald Reid, Aarhus University, Denmark Actinorhizal plants are able to establish a symbiotic relationship with Frankia bacteria Ton Bisseling, leading to the formation of root nodules. The symbiotic interaction starts with the Wageningen University & Research, Netherlands exchange of symbiotic signals in the soil between the plant and the bacteria. This *Correspondence: molecular dialog involves signaling molecules that are responsible for the specific Sergio Svistoonoff recognition of the plant host and its endosymbiont. Here we studied two factors [email protected] potentially involved in signaling between Frankia casuarinae and its actinorhizal host

Specialty section: Casuarina glauca: (1) the Root Hair Deforming Factor (CgRHDF) detected using a This article was submitted to test based on the characteristic deformation of C. glauca root hairs inoculated with Plant Evolution and Development, F. casuarinae and (2) a NIN activating factor (CgNINA) which is able to activate a section of the journal Frontiers in Plant Science the expression of CgNIN, a symbiotic gene expressed during preinfection stages Received: 31 May 2018 of root hair development. We showed that CgRHDF and CgNINA corresponded to Accepted: 25 September 2018 small thermoresistant molecules. Both factors were also hydrophilic and resistant to Published: 18 October 2018 a chitinase digestion indicating structural differences from rhizobial Nod factors (NFs) or Citation: Cissoko M, Hocher V, Gherbi H, mycorrhizal Myc-LCOs. We also investigated the presence of CgNINA and CgRHDF in Gully D, Carré-Mlouka A, Sane S, 16 Frankia strains representative of Frankia diversity. High levels of root hair deformation Pignoly S, Champion A, Ngom M, (RHD) and activation of ProCgNIN were detected for Casuarina-infective strains from Pujic P, Fournier P, Gtari M, Swanson E, Pesce C, Tisa LS, Sy MO clade Ic and closely related strains from clade Ia unable to nodulate C. glauca. Lower and Svistoonoff S (2018) Actinorhizal levels were present for distantly related strains belonging to clade III. No CgRHDF or Signaling Molecules: Frankia Root Hair Deforming Factor Shares CgNINA could be detected for Frankia coriariae (Clade II) or for uninfective strains from Properties With NIN Inducing Factor. clade IV. Front. Plant Sci. 9:1494. doi: 10.3389/fpls.2018.01494 Keywords: symbioses, nodulation factors, nodule inception, Casuarina, Alnus, Discaria

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INTRODUCTION Much less is known about signaling molecules involved in the actinorhizal symbioses. Canonical nodABC genes are not Legumes and actinorhizal plants form a N2-fixing root nodule found in the sequenced genomes of 36 Frankia strains including symbiosis in association with rhizobia and Frankia bacteria, Frankia alni and Frankia casuarinae (Tisa et al., 2016) confirming respectively (Vessey et al., 2005). The establishment of these a previous report showing that F. alni DNA will not complement beneficial bacterial-plant relationships requires communication rhizobial nod mutants (Cérémonie et al., 1998). Only distant between the partners. Rhizobial symbioses have received homologs of nodB and nodC are found in F. alni genome. Unlike considerable attention because several legumes are important rhizobial nod genes, they are not organized into a cluster together crop species. However, actinorhizal symbioses, which play an with other symbiotic genes and their expression is not induced important ecological role (Dawson, 2008), have been less well under symbiotic conditions (Normand et al., 2007; Alloisio studied and the molecular dialog between Frankia and their et al., 2010). These findings are consistent with experiments host plants is still poorly understood. One reason is that showing that chitin oligomers similar to rhizobial NFs are not be most actinorhizal plants are woody shrubs or trees for which detected in F. alni culture supernatant (Cérémonie et al., 1999) genetic approaches are very difficult (Wall, 2000; Perrine-Walker suggesting structural differences between the Frankia symbiotic et al., 2011). In addition, the genetics of the bacterial partner, signals and rhizobial NFs. Recently, canonical nodABC genes Frankia, is not fully developed and up to now Frankia cells have been found in the genome of two uncultured Frankia remain recalcitrant to stable genetic transformation (Kucho strains: Candidatus Frankia datiscae Dg1 and Candidatus Frankia et al., 2009, 2017). Recent progress including the sequencing californicae Dg2 (Persson et al., 2015; Nguyen et al., 2016), and of several Frankia genomes (Normand et al., 2007; Tisa in one isolated strain, Frankia sp. NRRL B-16219 (Ktari et al., et al., 2016), transcriptomic studies (Alloisio et al., 2010; 2017). F. datiscae Dg1 nodABC genes are arranged in two operons Benson et al., 2011), proteomic studies (Mastronunzio and which are expressed in Datisca glomerata nodules, but their Benson, 2010; Ktari et al., 2017) together with functional involvement in symbiotic signaling is still not known (Persson studies on several actinorhizal species (Svistoonoff et al., et al., 2015). 2014) have opened new avenues for identifying components Frankia is able to infect their host root either through involved in the initial symbiotic dialog between the two intracellular (root hair) or intercellular modes. In the first case, partners. one of the earliest visible plant response to Frankia is an extensive The interaction of rhizobia with model legumes begins deformation of root hairs. This response occurs in actinorhizal with the production and recognition of signal molecules by plants belonging to the order Fagales (Betulaceae, Casuarinaceae) their respective eukaryotic and prokaryotic symbiotic partners that display a range of relatively advanced features reminiscent of (Oldroyd, 2013). Early events leading to nodule formation model legumes: a complex root hair infection process involving involve bacterial penetration into their hosts via root hairs. the formation of ITs and the implication of cortical cell divisions Bacteria elicit the stimulation and reorientation of root hair at the initial stages of infection (Svistoonoff et al., 2014). Frankia cell wall growth. This rhizobia-induced tip growth results first culture supernatants also cause root hair deformation (RHD) and in the entrapment of the bacteria within curled root hairs and a Frankia root hair deforming factor in Alnus (AgRHDF) was then in the initiation and development of infection threads identified (Prin and Rougier, 1987; Ghelue et al., 1997; Cérémonie (ITs), tubular structures through which bacteria pass on their et al., 1999; Gabbarini and Wall, 2011). Using RHD as a bioassay, way down the root hair and into the underlying cortical cell partial purification was achieved. AgRHDF is a relatively small layers (Lhuissier et al., 2001). Ahead of the advancing threads, (< 3 kDa), heat stable, hydrophilic molecule that is resistant to a cells in the inner cortex are induced to dedifferentiate and chitinase treatment, but its chemical structure remains unknown divide, and a nodule primordium is formed. In the first part (Cérémonie et al., 1999). of the signal exchange, the plant roots secrete flavonoids that In recent years, we have developed complementary bioassays lead to the activation of a set of rhizobial genes (the nod using plant genes that are specifically expressed in response genes), which are essential for infection, nodule development to interaction with a compatible Frankia. This approach is and the control of host specificity (Masson-Boivin et al., 2009; particularly well suited for C. glauca where transgenic plants Oldroyd, 2013). These genes are responsible for the synthesis containing promoters of symbiotic genes fused to either GUS of lipo-chito-oligosaccharides (LCOs) called Nod factors (NFs) or GFP can be generated (Svistoonoff et al., 2010a). Expressed that signal back to the plant (Oldroyd, 2013). NF biosynthesis Sequence Tag (EST) libraries of C. glauca and Alnus glutinosa is dependent on nodABC genes which are present in all (Hocher et al., 2006, 2011) provide extensive lists of genes rhizobia able to synthetize NFs and strain-specific combinations potentially involved in the actinorhizal symbiosis. Among the of other nodulation genes responsible for the addition of candidate genes, we identified CgNIN, the putative ortholog various decorations to the core structure. (Masson-Boivin et al., of legume NIN genes, which encodes a transcription factor 2009). In model legumes, NFs perception elicits a range of playing a central role in rhizobial nodulation (Schauser et al., responses including ion fluxes, calcium oscillations, changes 1999; Marsh et al., 2007; Soyano et al., 2013, 2014; Yoro in gene expression patterns, and extensive deformation of et al., 2014). In C. glauca, CgNIN also has an important role roots hairs, which has been used as a bioassay to identify in nodulation particularly at early steps of infection (Clavijo the chemical nature of NFs (Lerouge et al., 1990; Oldroyd, et al., 2015). After contact with either Frankia cells or cell- 2013). free Frankia supernatants, the CgNIN promoter is strongly

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activated at 12 to 48 h (Clavijo et al., 2015). This property fluids were filtered through a 0.22 µm filter as described in was used to establish a new bioassay leading to the partial Chabaud et al., 2016. Unless otherwise indicated, experiments purification and characterization of a NIN activating factor, were performed with the supernatant fluids of a F. casuarinae called CgNINA. While rhizobial NFs are amphiphilic chitin- culture induced with RE from its host plant C. glauca and based molecules, CgNINA, like AgRHDF, is hydrophilic and referred to as FCS for Frankia casuarinae supernatant. FCS resistant to chitinase (Chabaud et al., 2016). However, it is not were concentrated fifty times (FCS 50X) using an R-210/215 known to what extent CgNINA is related to factors able to deform evaporator (BÜCHI Labortechnik AG, Switzerland). Nodulation root hairs. experiments with the different Frankia strains were performed Further experiments concerning these Frankia symbiotic as described previously (Alloisio et al., 2010; Svistoonoff et al., factors are reported here. We show that C. glauca was able to 2010b; Imanishi et al., 2011). perceive a root hair deforming factor secreted by F. casuarinae (CgRHDF) and the properties of CgRHDF were compared Characterization of F. casuarinae to those previously identified with CgNINA. The presence of Supernatant Fluids CgNINA and CgRHDF in strains representative of Frankia Temperature and pH Sensitivity diversity was investigated. To test heat inactivation, FCS 50X was autoclaved at 120◦C for 20 min. Cold sensitivity was determined by freezing FCS at - ◦ MATERIALS AND METHODS 80 C for 1 h. The effects of pH on FCS activity were determined as follows: the initial pH of FCS (6.7) was adjusted to pH 3, 5, Plant Material and Growth Conditions 7, 8 or 10 by adding either HCl or KOH solutions. The FCS mixtures that these pH values were incubated for 1h at room Casuarina glauca seeds (seed lot 15.934, ref.086-5929) were temperature and neutralized back to pH 6.7 by adding either provided by the Australian Tree Seed Centre1. Ochetophila HCl or KOH solutions before performing the bioassays described trinervis (= Discaria trinervis) seeds were collected from below. Samples that lost CgNINA or CgRHDF activities were plants growing in Pampa de Huenuleo (Bariloche, Argentina). sonicated for 30 min using a Branson 2510 sonicator. A. glutinosa seeds were harvested from a tree situated in the left bank of Rhône River in Lyon, France. C. glauca Size Fractionation and O. trinervis seeds were disinfected and germinated in a To estimate the size of signaling molecules, the FCS samples sterilized substrate for three weeks and transferred into glass were dialyzed as described in Chabaud et al., 2016. Ten mL tubes filled with a modified Broughton and Dilworth (BD) of FCS 50X were dialyzed for 12h at 4◦C against 5 L of medium as described previously (Ngom et al., 2016). A. glutinosa ultrapure water with stirring and using either a 100–500, 500– seeds were washed in distilled sterile water for 30 min before 1000 or 3500–5000 Da cutoff membrane (Float-A-Lyzer G2 sterilization in 96% ethanol for 30 min followed by 3% solution dialysis devices, Spectrum Laboratories, CA, United States). The of calcium hypochlorite for 30 min. Seeds were germinated dialyzed solutions were tested using the CgRHD and CgNINA ◦ on 1.5% plant agar for 10 days at 20 C, and transferred bioassays described below. The size of active compounds was in 5 mL tubes containing liquid Fahraeus medium without also estimated using centrifugal filters with cutoffs of 30, 10, and nitrogen (Fahraeus, 1957). Plants were grown for 6 weeks in 3 kDa (Amicon Ultra-4 centrifugal filters; Merck-Millipore, Cork, ◦ growth chamber at 25 C at 75% relative air humidity and Ireland). Four mL of 50X FCS were loaded on a 30 kDa cell 16 h light cycle/day. Transgenic C. glauca plants containing a which was spun at 4,000 g for 20 min. The filtrate recovered ProCgNIN:GFP construct described previously (Clavijo et al., from the 30 kDa filtration was treated similarly using a 10 kDa 2015) were grown in hydroponics in pots containing the modified cell, and the resulting 10 kDa filtrate was added to a 3 kDa Cell BD medium and vegetatively propagated as described previously and spun. (Svistoonoff et al., 2010b). Phase Extraction and Sensitivity to Chitinase Preparation of Cell-Free Supernatants Two sequential butanol extractions were performed on FCS 50X and Inoculation with a ratio 1-butanol / water (1:3; v/v) described previously (Chabaud et al., 2016). The butanol phase was evaporated at The bacterial strains used in this study are listed in ◦ Supplementary Table S1 and were grown for twenty-one 80 C under a nitrogen flow and the residue was dissolved in days in modified basic propionate (BAP) media described 20% acetonitrile as described in (Chabaud et al., 2016). Chitinase previously (Ngom et al., 2016) according to conditions listed digestions were performed on the aqueous phase extract as in Supplementary Table S1. Bacterial cultures were exposed to described previously (Chabaud et al., 2016). Chitinase activity plant root exudates (RE) for five days as described previously was assessed using a colorimetric method to estimate the amount (Beauchemin et al., 2012; Clavijo et al., 2015; Chabaud et al., of p-nitrophenol (p-NP) released from a reaction mixture 2016). Cell-free supernatant fluids were purified from cultures containing the substrate p-nitrophenyl N-acetyl glucosaminide showing an absorbance of 0.3 at 595 nm. Cultures were collected (p-NP-NAG) (Parham and Deng, 2000). A solution containing −1 by centrifugation at 4,000 g for 5 min and the supernatant 1 mg mL of Streptomyces griseus chitinase (C6137; Sigma- Aldrich) in a 50 mM phosphate buffer pH 6.0 was prepared. 1https://www.csiro.au/en/Research/Collections/ATSC A portion of this solution (100 µl) was mixed with 50 µl of 5X

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FCS aqueous extract, 100 µl of p-NP-NAG solution at10 mM Nikon) and a digital camera Sight D5 RI1 (Nikon). For each and 250 µl of acetate buffer (pH 5.5 0,1 M). The reaction was observation, a blind evaluation of GFP fluorescence levels incubated at 37◦C for 1 h under stirring and was terminated was performed using the following scale: 0: no detectable by the addition of 250 µl of CaCl2 at 0.5 M and 1000 µl of fluorescence; 1: weak fluorescence; 2: intermediate fluorescence; NaOH at 0.5 M. The amount of p-NP released was evaluated by and 3: strong fluorescence (Figure 1). For each experiment the measuring the absorbance at 400 nm with a spectrophotometer. number of plants with a given fluorescence level was used for Enzyme activity was expressed in µg of liberated p-NP per hour the statistical analysis described below. Each experiment was of incubation. Control reactions were performed without p-NP- repeated at least four times independently. NAG, without chitinase and without the aqueous FCS extract. Root Hair Deformation Bioassay in Root Hair Deformation and NINA A. glutinosa The deformation of A. glutinosa root hairs (AgRHD) was Bioassays in Casuarina glauca evaluated using 7 week-old plants that had at least four well Unless otherwise indicated, CgRHD and CgNINA bioassays were developed secondary roots. Biological tests were performed performed on aliquots of the same solution and the amount at 10−2 final dilutions of Frankia culture supernatant fluids −2 needed to achieve a final concentration equivalent a 10 dilution on plants growing in 5 ml Fahraeus media without nitrogen. of raw (no diluted) Frankia culture supernatant was added to the Evaluation of deformation was done in the region located about nitrogen-free BD medium. All experiments were performed on at 1.5 cm from the root tip and five levels of RHD were recorded least 4 plants. At least two independent experiments were carried for each observed root: 0a: no deformation; 0b: swelling; 1: out for each tested solution. branching; 2: branching and partial deformation; 3: total RHD The deformation of C. glauca root hairs (CgRHD) was and retracting. Deformation levels 0a and 0b were considered evaluated using 3 week-old non transgenic plants grown in as non-symbiotic. All experiments were performed on at least 3 glass tubes exposed to nitrogen starvation for one week as plants and 5 roots were observed for each plant. described previously (Ngom et al., 2016). Treatments were performed by replacing the medium with fresh nitrogen-free Statistical Analyses BD medium containing the assayed solution. Deformation of Statistical analyses were performed on raw data: the number of root hairs situated on small lateral roots was scored as described hairs counted in each level of deformation using the R software by Clavijo et al.(2015) using micrographs taken with a BX50F package (R Core Team, 2013). A Shapiro-Wilk normality test microscope (Olympus) equipped with a Micro Publisher 3.3 was performed followed by a non-parametric Kruskal-Wallis RTV (Qimaging) digital camera. A blind evaluation of each multiple comparison test and a pairwise Wilcoxon test. These micrograph was performed to determine the deformation level tests were used to compare the symbiotic response obtained for of observed root hairs using the following scale based on Clavijo each treatment. et al.(2015): 0a: no deformation; 0b: straight root hair with tip swelling; 1 weak deformation, only one change in growth Phylogenetic Tree direction; 2: intermediate deformation, more than one change in The strict core genome of 17 Frankia strains was determined with growth direction but no bifurcation; and 3: strong deformation: the Get_Homologs package (Contreras-Moreira and Vinuesa, one or more bifurcations (Figure 1). Deformation levels 0a and 2013). Out of 150,000 amino acid sequences in the Frankia pan 0b were considered non-symbiotic. For each experiment at least genome, 420 proteins were identified as orthologs and part of the four plants were analyzed per treatment and 6 small lateral roots strict Frankia core genome. A concatenated phylogenetic tree was were analyzed per plant. The total number of root hairs scored for constructed. These concatenations were aligned using Clustal W each level of symbiotic deformation was used for the statistical (Larkin et al., 2007). The distance matrix was computed by Jukes- analyzes described below. Each experiment was repeated four Cantor method (Jukes and Cantor, 1969). The Neighboring- times independently. joining method (Saitou and Nei, 1987) was used to build For each treatment the percentage of symbiotic deformation the phylogeny. The percentage of replicate trees in which (%SyD) defined as the proportion of root hairs showing a the associated taxa clustered together was determined using a symbiotic response was calculated and used to determine a bootstrap test (1000 replicates) (Felsenstein, 1985). Streptomyces deformation index using the following scale: level 1: SyD < 15%; coelicolor was used as an outgroup. level 2: 16% < SyD < 25%; level 3: 26% < SyD < 40%; level 4: SyD > 41%. The activation of ProCgNIN in response to tested solutions RESULTS was evaluated using transgenic ProCgNIN:GFP plants that were grown in hydroponics deprived of nitrogen for one week as CgRHD and CgNINA Activities Are previously described (Clavijo et al., 2015; Chabaud et al., 2016). After 24 h of contact with tested solutions, GFP fluorescence Present in F. casuarinae Supernatant was monitored in the short lateral root hairs using an AZ100 Fluid macroscope (Nikon) equipped with a 5X objective, a GFP filter Previous studies have shown that factors inducing RHD in (Excitation filter 470 nm ± 40 nm; Barrier filter 535 nm ± 50 nm; A. glutinosa (AgRHDF) are present in several Alnus-infective

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FIGURE 1 | Bioassays used to quantify the activation of ProCgNIN and root hair deformation in C. glauca. (A–D) Representative images showing levels of GFP fluorescence in root hairs of transgenic ProCgNIN:GFP C. glauca plants used for the CgNINA bioassay. Arrowheads indicate root hairs. (A) no signal, level 0; (B) weak signal, level 1; (C) medium signal, level 2; (D) strong signal, level 3. (E–I) representative images showing levels of root hair deformation in C. glauca used in the CgRHDF bioassay. (E) no deformation, level 0a; (F) tip swelling, level 0b; (G) one change in growth direction, level 1; (H) more than one change in growth direction but no bifurcation;, level 2; (I) one or more bifurcations, level 3. Arrowheads indicate deformed root hairs.

Frankia strains (Prin and Rougier, 1987; Ghelue et al., 1997; the CgNINA bioassay is more sensitive that the one based on Cérémonie et al., 1999). We recently found that F. casuarinae CgRHD. The last three dilutions (10−2 to 10−4) appear to show produces an extracellular factor, named CgNINA, which is able dose-dependent responses for the CgNINA bioassay that may be to induce the expression of the early symbiotic gene CgNIN in used to quantify this factor. small lateral roots (Clavijo et al., 2015; Chabaud et al., 2016). To investigate whether a root hair deforming factor, hereafter named CgRHDF, was also produced by F. casuarinae, we incubated wild CgRHDF and CgNINA Share type C. glauca plants with 2 10−1, 10−2 10−3, and 10−4-fold Physio-Chemical Properties dilutions of F. casuarinae supernatant fluids (FCS) and scored CgRHDF and CgNINA properties were further compared by the deformation of root hairs situated in small lateral roots. using similar treatments to those used to characterize CgNINA In parallel, transgenic plants containing the ProCgNIN:GFP (Chabaud et al., 2016). First, the effects of temperature and construct were incubated with the same solutions and the pH sensitivity were analyzed. As shown in Figures 3A,B and activation of ProCgNIN was recorded. As shown in Figure 2 Supplementary Table S2, CgRHD was not affected by elevated and Supplementary Table S2, no RHD and no GFP fluorescence temperatures (autoclaving) or treatment at pH values ranging were detected in negative control roots treated with diluted BAP from 5 to 10. However, cold treatment (freezing) or acidic − medium. RHD and GFP expression were maximal for the 10 2 pH conditions severely decreased CgRHD levels. Sonication dilution and lower levels of deformation and GFP fluorescence of the inactivated fractions (frozen or acid-treated) resulted was observed with higher dilutions. The more concentrated in a partial recovery of CgRHD activity (Figures 3A,B and dilution had a decreased response for both RHD and GFP Supplementary Table S2). Similar results were obtained with fluorescence suggesting that the receptor could be saturated or the CgNINA bioassay. These results suggesting that both factors inhibitory compounds may be associated with the extracts. At the are thermoresistant and possibly precipitate at low pH or upon − − dilutions 10 1 and 10 4, only the CgNINA bioassay showed a freezing but this aggregate can be resuspended using sonication. significant difference with the negative control suggesting that Both dialysis membranes and centrifugal filters were used to

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Presence of RHDF and CgNINA in Frankia Strains Representative of Frankia Diversity We were interested in determining whether other Frankia strains had CgNINA and RHDF activities and tested different strains representing Frankia diversity. Cell-free supernatant fluids corresponding to 16 Frankia strains and another Actinobacteria, S. coelicolor, were tested for their capacity to deform C. glauca root hairs. As shown in Figure 4, Supplementary Table S3, and Supplementary Figure S2, CgRHD was present in all the Frankia belonging to clades I and III. The strongest activities were detected in strains that nodulate the genus Casuarina (clade Ic). Remarkably, RHD activity was also detected for several Frankia strains unable to form nodules on Casuarina such as F. alni (Clade Ia, Alnus-infective), Frankia eleagni and EANIpec

FIGURE 2 | Effect of cell-free F. casuarinae supernatant fluids (FCS) on (both clade III, Elaeagnus-infective strains). No deformation was Casuarina glauca root hair deformation (CgRHDF bioassay) and the activation detected for Frankia coriariae (clade II). Supernatant fluids from of ProCgNIN (CgNINA bioassay). Plants were incubated with Frankia culture the atypical strains from clade IV or from the non-Frankia supernatant fluids (FCS) at the indicated dilutions and the Frankia culture actinobacterium S. coelicolor did not induce RHD on Casuarina. medium BAP was used as a negative control. Orange bars represent the Similar results were obtained when the same supernatant fluids proportion of deformed root hairs in short lateral roots 2 days after contact with FCS dilutions. Green bars represent the proportion of plants expressing were tested with the NIN bioassay (Figure 4, Supplementary GFP in short lateral roots at different levels. Asterisks above bars indicate Table S3, and Supplementary Figure S2) except for F. discariae. symbiotic responses significantly different from the negative control (P < 5%). The strong RHD and NINA activities obtained with the Alnus- strain F. alni prompted us to investigate whether the same culture supernatant fluids could induce RHD on A. glutinosa. As shown determine the approximate size of CgRHDF and CgNINA. As in Figure 4 and Supplementary Figure S2, responses observed shown in Figure 3C and Supplementary Table S2, CgRHDF in A. glutinosa were generally similar or stronger compared to and CgNINA were detected inside the 100–500 Da and the 500- the ones recorded in C. glauca particularly for strains belonging 1000 Da cut-off dialysis tubing, but only a residual activity was to group III. Interestingly, strains that were not able to activate found in the 3.5–5 kDa) suggesting that both factors correspond ProCgNIN or to deform root hairs of C. glauca such as F. coriariae to small molecules with a molecular mass between 1 and 3.5 kDa. and the two atypical strains from group IV, EuI1c and CN3 were Experiments performed using centrifugal filters with 30, 10, able to deform A. glutinosa root hairs. To confirm the absence of and 3 kDa cut-offs yielded similar results for the CgNINA unintentional contaminations with a compatible strain, C. glauca, bioassay (Figure 3D and Supplementary Table S2). However, A. glutinosa, and O. trinervis were inoculated with the bacterial maximum activity using the CgRHD bioassay was detected in pellet obtained while preparing the supernatant fluids described the 3 kDa and 10 kDa retentates and only residual CgRHD was above. As shown in Supplementary Table S3, nodules were detected in the 3kDa flow though suggesting that both factors only obtained with compatible strains, thus demonstrating that correspond to small but distinct molecules. Taken together these responses recorded with incompatible strains are not the result of experiments suggest that the size of CgNINA is between 1 and any contamination; incompatible strains are thus probably able 3 kDa while CgRHDF is between 3 and 3.5 kDa. The polarity to synthetize CgRHDF and CgNINA. of the two factors was investigated using a butanol extraction. Surprisingly for F. discariae, we detected not only weak levels CgRHDF and CgNINA were only detected in the aqueous of RHD in C. glauca but also a weak activation of ProCgNIN phase, indicating that both factors are hydrophilic (Figure 3E and a strong deformation of root hairs in A. glutinosa (Figure 4 and Supplementary Table S2). Finally, we investigated whether and Supplementary Figure S2). These results are in apparent CgRHDF contains a chitin backbone by performing the chitinase contradiction with our previous work, in which we could not digestion experiment described in Chabaud et al.(2016). As detect any activation of ProCgNIN in response to F. discariae shown in Figure 3F and Supplementary Table S2, the incubation supernatants. However, at that time F. discariae cultures were with chitinase had no significant effect on CgRHDF or CgNINA induced with RE from O. trinervis instead of C. glauca (Chabaud activities. To rule out any inhibitory effect by FCS, we quantified et al., 2016). We therefore repeated the experiments for F. alni the chitinase activity in the FCS/chitinase solution. As shown in and F. discariae using RE from A. glutinosa and O. trinervis, Supplementary Figure S1, chitinase activity was not decreased respectively. As shown in Figure 4, Supplementary Table S3, by the addition of FCS to the chitinase solution. We conclude that and Supplementary Figure S2, incubation of F. alni with RE F. casuarinae secretes two factors, CgNINA and CgRHDF, which from the host plant (A. glutinosa) did not change the response are possibly two distinct molecules. Both factors sharing similar of C. glauca, but slightly increased the response of A. glutinosa. biochemical properties and correspond to small hydrophilic and For F. discariae, incubation with exudates from O. trinervis thermoresistant molecules lacking a chitin backbone. reduced the level of ProCgNIN activation compared to the

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FIGURE 3 | Physico-chemical properties of CgRHDF and CgNINA. Orange bars show the proportion of deformed root hairs in short lateral roots 2 days after contact with FCS submitted to the different treatments. BAP medium diluted 100 times was used as a negative control. Asterisks above bars indicate symbiotic responses significantly different from the negative control (P < 5%). (A) Temperature sensitivity. High levels of CgRHD and GFP were detected in autoclaved FCS but no significant activity was present in FCS that was previously frozen. Sonication of frozen FCS (Frozen-S) allowed partial recovery of CgRHD and CgNINA activities. (B) pH sensitivity: FCS were incubated at different pH for one hour at the indicated pH. Sonication of FCS incubated at pH3 (pH3-S) restored CgRHD and CgNINA levels similar to the untreated control. (C) Size estimation using a dialysis tubing. CgRHD and CgNINA activities inside dialysis tubings with the indicated cutoffs was scored (D) Size estimation using centrifugal filters. FCS were submitted to successive filtrations using filters with the indicated cut-offs. CgRHD and CgNINA were evaluated on the retentate or the flow through (Flow t.). (E) CgRHD and CgNINA activity after 1-butanol extraction. Significant activities were only detected in the aqueous fraction (FCSaq) and not in the organic fraction (FCSorg). (F) Sensitivity to Chitinase. FCSaq incubated with chitinase showed similar CgRHD and CgNINA activities compared to untreated FCS.

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FIGURE 4 | Distribution of CgRHD, CgNINA, and AgRHD activities in Frankia strains representative of Frankia diversity. A genome-wide phylogenetic tree of 17 Frankia strains and S. coelicolor was constructed using the concatenated sequences of 420 shared proteins. Distances were computed by the Jukes-Cantor method and the neighbor-joining method was used to build the tree. One thousand bootstrap replications were used to evaluate statistical support for branches. Cell-free supernatant fluids of each strain were tested for their ability to induce RHD and the activation of ProCgNIN on C. glauca and RHD on A. glutinosa. Levels of response recorded for each strain correspond to the maximum deformation index and the maximum GFP fluorescence level for C. glauca and the maximum deformation level for A. glutinosa.

experiment performed with RE from C. glauca or without RE inoculation and factors able to induce RHD in Alnus (AgRHDF) and the results obtained were not significantly different from have been partially purified and characterized (Cérémonie the negative control (Supplementary Figure S2). Together, these et al., 1999). In C. glauca, we have used transgenic plants results showed that F. discariae is able to synthetize molecules expressing a ProCgNIN:GFP fusion to characterize CgNINA, able to induce CgRHD, AgRHD, and the activation of ProCgNIN. a factor present in cell free F. casuarinae supernatant fluids The activation of ProCgNIN was enhanced when F. discariae able to activate the CgNIN promoter in C. glauca root hairs. was incubated with RE from C. glauca and was possibly below Here we have shown that a CgRHDF is also present in cell- detection limits using the less sensitive equipment described in free F. casuarinae supernatant fluids. Furthermore, we have (Chabaud et al., 2016). shown that CgRHDF and CgNINA share similar physico- chemical properties listed in Table 1. Interestingly the experiment performed with centrifugal filters suggests that CgNINA and DISCUSSION CgRHDF are both small molecules with slightly different sizes, CgNINA being probably smaller than CgRHDF. This observation Presence of CgRHDF and Comparison to is intriguing if CgNINA or CgRHDF are the actinorhizal analogs CgNINA of rhizobial NFs because NFs are known to induce both RHD In legumes, RHD is one of the earliest visible responses induced and the expression of early nodulins such as NIN. Responses upon recognition of rhizobial NFs by the host plant, and obtained with centrifugal filters were however, less contrasted the development of a bioassay based on RHD was crucial to compared to the other experiments shown here and residual identify the chemical nature of NFs (Lerouge et al., 1990). CgRHD activity was still present in the 3kDa flow through. We The perception of NFs also provokes significant alterations therefore cannot exclude that CgNINA also possess a small RHD of gene expression and notably the expression of symbiosis- activity and additional experiments are needed to confirm this induced genes such as MtEnod11 (Journet et al., 2001) and NIN hypothesis. If CgRHDF and CgNINA can indeed be separated, (Schauser et al., 1999; Radutoiu et al., 2003). In actinorhizal it would be interesting to know if those molecules are able to + plants infected intracellularly such as C. glauca and A. glutinosa, induce the high frequency nuclear Ca2 spiking in growing RHD is also one of the first visible responses to Frankia C. glauca root hairs as described previously (Chabaud et al.,

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TABLE 1 | Properties of CgRHDF, CgNINA, AgRHDF, and rhizobial NFs.

CgRHDF CgNINA AgRHDF Rhizobial NF

Induction Inducible Inducible Inducible Inducible (root exudates) (root exudates) (root exudates) (flavonoids) Size 1–3 kDa 1–3 kDa 1.2–3 kDa 1 kDa Thermal stability Thermoresistant Thermoresistant Thermoresistant Thermoresistant Active concentration 10−2–10−3 (supernatant) 10−1–10−4 (supernatant) 10−3 10−5 (supernatant) Down to 10−12 M Hydrophilicity Hydrophilic Hydrophilic Hydrophilic Amphiphilic Chitinase action resistant resistant resistant sensitive

2016). The ability to induce Ca2 + oscillations in response CgRHDF/NINA or AgRHDF, even if their chemical backbone is to symbiotic bacteria is a common feature of nodulating not chitin-based. species within the nitrogen-fixing clade (Granqvist et al., 2015). Alternatively we hypothesize that CgRHDF and CgNINA are Presence of NINA and CgRHDF in Other the same molecule but a cofactor or a specific decoration is Frankia Strains needed to enhance CgRHD activity without affecting its ability In most legumes, NFs allow the specific recognition between to activate ProCgNIN. The 3 kDa centrifugal filter possibly the host plant and its symbiotic rhizobia (Masson-Boivin et al., eliminated decorated molecules with higher mass or cofactors 2009; Oldroyd, 2013). Changes in specific decorations often and therefore strong activity was only detected with the CgNINA result in host incompatibility (Dénarié et al., 1996) but NFs bioassay. from incompatible strains can induce symbiotic responses such Comparison With AgRHDF and Rhizobial as RHD and activation of symbiotic genes when applied at increased concentrations (Roche et al., 1991). These results can Nod Factors be explained by changes of affinity between NFs and the cognate CgRHDF and CgNINA also share many characteristics NF receptors able to recognize the chitin backbone and also with AgRHDF, the corresponding factor characterized using the modified backbone structure. A misrecognition leads to A. glutinosa/F. alni (Table 1; Ghelue et al., 1997; Cérémonie decreased affinity but this can be compensated by increased et al., 1999). However, both factors appear to be structurally amounts of substrate (Dénarié et al., 1996). The symbiotic different from the rhizobial NFs because unlike NFs they are responses in non-host plants reported here point to a similar not found in the organic phase following a butanol extraction mechanism in C. glauca: strains from clades I and III possibly and are not sensitive to the endochitinase from Aeromonas synthesize molecules sharing a common molecular backbone hydrophila (Cérémonie et al., 1999) or the exochitinase from that is recognized by C. glauca receptors inducing RHD and S. griseus (Cérémonie et al., 1999; Chabaud et al., 2016). the activation of ProCgNIN promoter. Optimal recognition is This difference is in agreement with (1) the lack of nodA achieved for compatible strains (clade Ic) and some related strains genes in the sequenced genomes of F. casuarinae and F. alni (F. alni from clade Ia) but only the backbone would be recognized (Normand et al., 2007). (2) the absence of chitin oligomers for more distant strains (clade III). This recognition is not in F. alni supernatant fluids (Cérémonie et al., 1999), and detectable for non-infective strains (clade IV) and the distantly (3) the failure of NFs from the broad host-range rhizobia 2 + related strain F. coriariae suggesting that those strains do not NGR234 to elicit RHD or Ca spiking in A. glutinosa or produce sufficient amounts of this recognized backbone under C. glauca (Cérémonie et al., 1999; Granqvist et al., 2015; Chabaud the tested conditions. et al., 2016). The possibility that actinorhizal recognition is mediated by molecules that are not hydrolyzed by tested chitinases is unexpected because downstream components of Comparison Between CgRHDF and the NF signaling pathway are conserved not only between AgRHDF actinorhizal and rhizobial symbioses (Svistoonoff et al., 2014; Compared to CgRHDF and CgNINA, the distribution of Griesmann et al., 2018), but also between rhizobial and AgRHDF seems less related to phylogeny. Generally, AgRHDF arbuscular-mycorrhizal symbioses where chitin-derived Myc- levels were stronger for clade III strains and several strains LCOs and COs play an important role as signaling molecules without any CgRHDF or CgNINA activity (F. coriariae and two (Camps et al., 2015). Putative orthologs of NF receptors are uninfective strains from clade IV). These differences suggest that present in C. glauca and A. glutinosa (Hocher et al., 2011). the AgRHDF assay detects smaller concentrations of deforming We are currently studying whether these genes play a role in factors or that root hairs of A. glutinosa are deformed by a actinorhizal symbioses. Because LysM receptor kinases have wider range of molecules compared to C. glauca. This second been shown to recognize not only chitin-derived molecules hypothesis is in agreement with RHD detected in Alnus roots but also peptidoglycans and exopolysaccharides (Willmann incubated with non-Frankia bacteria or fungi (Berry and Torrey, et al., 2011; Kawaharada et al., 2015), orthologs of genes 1983; Knowlton and Dawson, 1983; Prin and Rougier, 1987; encoding NF receptors could be involved in the recognition of Sequerra et al., 1994).

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Impact of Root Exudates experiments. MC, SSv, PP, PF, and VH analyzed the data. SS We also found that the nature of RE used to incubate Frankia and MC statistical analyzed the data. MS, MG, MC, VH, HG, cultures could have an impact on RHDF and CgNINA activities. DG, AC-M, SP, AC, MN, ES, CP, and SSv contributed reagents, Unexpectedly lower CgRHDF and CgNINA activities were found materials, analysis, and tools. MC, SSv, and LT wrote the paper. when F. discariae was cultivated with RE from O. trinervis All authors read the final version of the manuscript. compared to RE from C. glauca. In legumes, specific flavonoids secreted by the host plant induce the expression of nod genes and the synthesis of NFs (Oldroyd, 2013). RE secreted by the FUNDING host plant probably also play a role in actinorhizae formation because the incubation with RE induces morphological changes This work was supported by IRD (French National Research in Frankia and accelerate the nodulation process (Gabbarini and Institute for Sustainable Development), and the United States Wall, 2008, 2011; Beauchemin et al., 2012). Myricaceae seed Department of Agriculture (project USDA NIFA 2015-67014- extracts also influence Frankia growth (Bagnarol et al., 2007). In 22849). Alnus AgRHDF is reported to be produced either constitutively (McEwan et al., 1992; Ghelue et al., 1997; Cérémonie et al., 1999) or upon induction with RE (Prin and Rougier, 1987). ACKNOWLEDGMENTS Different plant RE have different effects on Frankia physiology. Information about CgRHDF is scarce but flavonoids isolated We would like to thank Maurice Sagna (UCAD, Dakar) for from Casuarina seeds have been shown to induce the production his help with FCS concentration, L. Wall (Quilmes University, of CgRHDF by the Casuarina-infective BR strain (Selim, 1995). Argentina) and E. Chaia (U. Comahue, Argentina) for providing Increased CgRHD activity in F. discariae incubated with C. glauca O. trinervis seeds. RE could be due to increased amounts of flavonoids in Casuarina RE compared to O. trinervis. SUPPLEMENTARY MATERIAL AUTHOR CONTRIBUTIONS The Supplementary Material for this article can be found online MC, VH, PP, MS, and SSv conceived and designed the at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01494/ experiments. MC, PP, PF, VH, and AC-M performed the full#supplementary-material

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Cissoko et al. Properties of Actinorhizal Signaling Molecules

Svistoonoff, S., Hocher, V., and Gherbi, H. (2014). Actinorhizal root nodule Yoro, E., Suzaki, T., Toyokura, K., Miyazawa, H., Fukaki, H., and Kawaguchi, M. symbioses: what is signalling telling on the origins of nodulation? Curr. Opin. (2014). a positive regulator of nodule organogenesis, nodule inception, acts as a Plant Biol. 20, 11–18. doi: 10.1016/j.pbi.2014.03.001 negative regulator of rhizobial infection in Lotus japonicus. Plant Physiol. 165, Tisa, L. S., Oshone, R., Sarkar, I., Ktari, A., Sen, A., and Gtari, M. (2016). Genomic 747–758. doi: 10.1104/pp.113.233379 approaches toward understanding the actinorhizal symbiosis: an update on the status of the Frankia genomes. Symbiosis 70, 5–16. doi: 10.1007/s13199-016- Conflict of Interest Statement: The authors declare that the research was 0390-2 conducted in the absence of any commercial or financial relationships that could Vessey, K. J., Pawlowski, K., and Bergman, B. (2005). Root-based N 2- be construed as a potential conflict of interest. fixing symbioses: legumes, actinorhizal plants, Parasponia sp. and cycads. Root Physiol. Gene Funct. 266, 51–78. doi: 10.1007/1-4020- Copyright © 2018 Cissoko, Hocher, Gherbi, Gully, Carré-Mlouka, Sane, Pignoly, 4099-7_3 Champion, Ngom, Pujic, Fournier, Gtari, Swanson, Pesce, Tisa, Sy and Svistoonoff. Wall, L. G. (2000). The actinorhizal symbiosis. J. Plant Growth Regul. 19, 167–182. This is an open-access article distributed under the terms of the Creative Commons Willmann, R., Lajunen, H. M., Erbs, G., Newman, M.-A., Kolb, D., Tsuda, K., et al. Attribution License (CC BY). The use, distribution or reproduction in other forums (2011). Arabidopsis lysin-motif proteins LYM1 LYM3 CERK1 mediate is permitted, provided the original author(s) and the copyright owner(s) are credited bacterial peptidoglycan sensing and immunity to bacterial infection. and that the original publication in this journal is cited, in accordance with accepted Proc. Natl. Acad. Sci. U.S.A. 108, 19824–19829. doi: 10.1073/pnas.11128 academic practice. No use, distribution or reproduction is permitted which does not 62108 comply with these terms.

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